Proteomic Analysis of eIF5B Silencing-Modulated Proteostasis

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Protein translation is crucial for cellular proteostasis and aging.
• Inhibiting translation initiation factors boosts stress resistance in model organisms.
• Eukaryotic translation initiation factor 5B (eIF5B) is key in forming the 80S ribosomal complex during translation initiation.

Purpose of the Study:

• To investigate the physiological effects of altered eIF5B expression in human cells.
• To understand how eIF5B levels impact proteostasis, stress responses, and cell growth.

Main Methods:

• eIF5B knockdown (eIF5B-KN) in 293T and HepG2 cells.
• Proteomic analysis to identify differentially expressed proteins.
• Western blotting and qPCR to confirm signaling pathway alterations.
• Assessment of reactive oxygen species (ROS), oxidative stress resistance, and autophagy.

Main Results:

• eIF5B-KN cells exhibited slower growth, reduced ROS, increased oxidative stress resistance, and enhanced autophagy.
• Proteomic analysis revealed significant changes in protein expression related to metabolism, RNA processing, and DNA synthesis.
• Autonomous downregulation of the MAPK signaling pathway and inactivation of the mTOR pathway were observed.
• Decreased 28S rRNA and 5.8S rRNA levels were noted, which were reversible upon eIF5B restoration.

Conclusions:

• eIF5B knockdown triggers a negative feedback loop that deactivates MAPK signaling, leading to reduced cell growth.
• Altered eIF5B expression impacts proteostasis by modulating MAPK signaling, cell cycle progression, and autophagy.
• These findings highlight the role of protein synthesis in regulating cellular stress responses and proteostasis.